Abstract 5986: The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An <i>in vitro</i> and <i>in vivo</i> study
Authors: Nitisha Shrivastava, Nicole Kawachi, Michael B. Prystowsky, Evripidis Gavathiotis, Chandan Guha, Thomas J. Ow
Journal: Cancer Research (2023)

Abstract INTRODUCTION: CDKN2A, coding for p16INK4a, a CDK4/6 inhibitory protein, is among the most commonly lost tumor suppressors in oral cavity squamous cell carcinoma (OCSCC). Drugs like palbociclib are CDK4/6 inhibitors that phenomenally also induce senescence. Senescence can be pro-tumorigenic. Studies suggests prosurvival BCL-2 family support tumor cell survival in senescent state. Agents targeting BCL-2 family e.g. navitoclax, are “senolytics” The current study examines combining palbociclib (P) with the senolytic agent navitoclax (N) as a treatment strategy. We demonstrate efficacy of combining P+N in OCSCC, and examine senescence and apoptosis processes as related mechanistic response of combination. EXPERIMENTAL PROCEDURE: We examined cell viability after sequential treatment: 72 h P followed by 72 h N (P+N) in 2-dimensional (2D) model of immortalized cell line HN5 and 3D model of a human OCSCC patient-derived conditional reprogrammed cell line (CR18). Flow cytometry measured apoptosis with Annexin V assay in 2D. Confocal imaging in 3D spheroids corroborated pro-survival protein levels. An in vivo xenograft study was performed with HN5 in 8 weeks old male nude mice to examine tumor responses to sequential P+N treatment. 3 million cells were injected in right flank. Tumors were treated at ~300 mm3 (P-100 mg/kg o.p; N -17 mg/kg i.p). After treatment termination, Bcl-2 family proteins (BcLxl); apoptosis markers (caspase 3, Cleaved PARP) and β-galactosidase (β -gal) for senescence were evaluated with western blot. QPCR measured expression of CDK4/6 target (FOXM1) to examine senescence and pro survival genes. RESULTS: Sequential treatment of P+N (1µM each) demonstrated consistent synergy in both 2D and 3D viability assays. Bright field images of P+N revealed senescence with P followed with apoptosis on N application. β -gal levels increased to 1.5 fold with P and Caspase 3 showed 2-fold increase with P+N. Flow cytometry data revealed 2 folds increase in the apoptotic population with P+N. Confocal imaging in organoids demonstrated increased levels of BcLxL with P, and repression of BCL-xL following N administration. Sequential P+N significantly reduced tumor growth in vivo. P+N demonstrated a robust 5-fold decrease in tumor volume (TV) with a complete cure (TV&amp;lt;20 mm3) in 60% population in comparison to either of the cohorts post 30 days of treatment (control: 2000 mm3, P- 1200 mm3; N- 1500 mm3, p&amp;lt;0.05). Compared to control sequential P+N treatment in vivo i) reduced anti-apoptotic BcLxL mRNA expression to 2.5 fold ii) Cleaved PARP increased 10 folds and iii) BcLxL levels decreased 2 fold. CONCLUSION: Our results suggest that CDK4/6 inhibition makes OCSCC undergo senescence and make it more susceptible to apoptosis when combined with the BCL2-family inhibitor, navitoclax. We propose a sequential P+N as a rational and potentially effective treatment strategy in OCSCC. Citation Format: Nitisha Shrivastava, Nicole Kawachi, Michael B. Prystowsky, Evripidis Gavathiotis, Chandan Guha, Thomas J. Ow. The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5986.